The binding of proteins from an immortalised B-cell line to a panel of SH3 domains was investigated in vitro. One of the most prominent SH3 domain binding proteins was a 68 kD polypeptide which strongly associated with the SH3 domains of c-src, p85a and p47phox and weakly with the SH3 domain of PLCgamma and n-src with undetectable binding to the other SH3 domains tested. Immunoblotting identified this protein as human Sam68. The ability of proline-rich peptides homologous to the Sam68 primary sequence to inhibit the binding of Sam68 to SH3 domains was investigated. Only one peptide inhibited binding of Sam68 to the p85alpha SH3 domain, whereas several peptides inhibited binding of Sam68 to c-src SH3 domain, suggesting that Sam68 uses different proline-rich motifs to bind to different SH3 domains. A peptide derived from residues 32-44 of Sam68 which fits the class II SH3 domain binding consensus sequence inhibited binding of Sam68 to both p85alpha SH3 domain and c-src SH3 domain, but with differential potency, suggesting a differential affinity of these SH3 domains for this proline-rich motif.